PROJECT SUMMARY Protein synthesis by the ribosome is central to life and disease. We have developed single- molecule approaches to provide a real-time dynamic perspective to the basic and unusual steps of protein synthesis. Here we build on our methodological developments over the past funding period to provide combined structural and dynamic view of how mRNA and nascent protein sequence and structure modulate translation and affect its outcome. In Aim 1 we focus initiation and termination. We will understand the conformational dynamics of the 30S subunit during initiation and how the process is controlled by mRNA sequence and structure; we will study termination regulation by RF3, and recoding in selenocysteine incorporation; finally, we will watch how polysomes form. In Aim 2, we will study the role of mRNA sequence and structure in controlling elongation. We will investigate how modified nucleotides in mRNA change elongation, and study how ribosomes navigate secondary structures. We will determine the basis for pausing and reading-frame changes that occur during elongation due to mRNA sequence or structure, tracking pathways, branchpoints and mechanism. We will also determine how two ribosomes translating on an RNA navigate secondary structures and possibly interact during elongation. In Aim 3, we explore the interplay of nascent protein sequence and folding with elongation kinetics. We will determine how antibiotic sensitivity or resistance is determined by the nascent chain, and how translation is disrupted. We will determine how the folding of well-characterized protein domains on the ribosome affect real time translation (pausing, stalling acceleration). Finally, we will explore the directly the correlation of folding and translation in real time. The proposed research is buttressed by strong collaborations on the various systems to support biophysical and structural analysis, reagent preparation, and in vivo correlation. The results of this proposal will provide a deeply textured view for how protein synthesis is regulated and controlled.